Polysaccharide-polypeptide conjugate

ABSTRACT

Disclosed is a method of producing a polysaccharide-polypeptide conjugate by reacting a polysaccharide with a polypeptide which contains at least one free amino group, wherein a polysaccharide carrier comprising vicinal hydroxyl groups is oxidized under ring opening to create vicinal aldehyde groups and is reacted with one or more base-instable antigenic polypeptide(s) containing at least one free amino group, the polypeptide(s) being bound directly to the polysaccharide carrier via at least one azomethine bond.

The present invention relates to a new use of oxidized polysaccharidesas a carrier material for components of vaccines, in particular to amethod of producing a polysaccharide-polypeptide conjugate by reacting apolysaccharide with a polypeptide comprising at least one free aminogroup, as well as to the use of such a conjugate as a vaccine.

Vaccines are characterized in that one or more antigens are administeredin an immunogenic formulation in a small amount, mostly parenteral(subcutaneously or intramuscularly) so as to trigger a strong andprotective immune response. At present, most vaccines are produced forprotecting against microbial infections. In these instances, theantigens used are inactivated and altered microorganisms or partsthereof, or defined proteins from such microorganisms which are suitableto trigger an immune response against the respective microorganism.

For years also the effectiveness of many experimental vaccines againstother diseases has been investigated. Among them are vaccines againstcancer. In this case, the immune system of cancer patients is to beselectively activated so as to combat malignant cells. This is attemptedby means of the most differing approaches. Among them are vaccinationswith autologous or allogenic tumor cells, chemically ormolecular-biologically modified autologous or allogenic tumor cells,isolated tumor-associated antigens (TAA) or tumor-associated antigensprepared by chemical or molecular-biological methods, peptides derivedtherefrom, anti-idiotypical antibodies as a surrogate of a TAA, latelyalso vaccinations with DNA which codes for TAA or for structures derivedtherefrom, etc. In principle, very small amounts of a suitable vaccinewill suffice to induce an immunity from months up to years, since theattenuation can be boosted by booster vaccinations. Moreover, in anactive immunization both a humoral and a cellular immunity can beinduced the interaction of which can yield an effective protectionagainst cancer.

To attain a strong immunity, antigens in vaccines mostly areadministered together with an adjuvant. As examples of adjuvants thefollowing may be mentioned, without, however, being restricted thereto:aluminum hydroxide (Alu-Gel), derivatives of lipopolysaccharide,Bacillus Calmette Guerin (BCG), liposome preparations, formulations withadditional antigens against which the immune system has already produceda pronounced immune response, such as, e.g., tetanus toxoid, Pseudomonasexotoxin or components of influenza viruses, optionally in a liposomepreparation. Furthermore, it is known that the immune response may alsobe enhanced by simultaneously administering endogenous proteins whichplay an important role in the build-up of an immune response, such as,e.g., granulocyte macrophages-stimulating factor (GM-CSF), interleukin 2(IL-2), interleukin 12 (IL-12) or gamma interferon (IFNγ).

U.S. Pat. No. 5,554,730-B relates to polysaccharide-protein conjugates,wherein a particulate vaccine is to be created. For this purpose, apolysaccharide-protein conjugate is created as a Schiff's base(azomethin), primarily by reacting a protein carrier with an oxidizedpolysaccharide antigen in the presence of a “crowding agent” (waterdisplacing agent), wherein the protein carrier is immediately denatureddue to the presence of the crowding agent, and the conjugateprecipitates in the form of microparticles. Although a dissolution ofthe precipitated microparticles in a strongly basic environment (0.1 NNaOH) for obtaining a vaccination solution as such is possible and hasalso been disclosed, it only makes sense if a polysaccharide antigen isused, because any antigenic protein would have lost its antigenicdeterminants as a consequence of denaturing, and thus would no longer beeffective.

WO 99/55715 describes polysaccharide-antigen conjugates in which theantigen is either bound to the polysaccharide via a suitable bivalentlinker, or via a terminal aldehyde group. A direct binding of theantigen to the polysaccharide via an azomethin bond thus is limited tothe number of the terminal aldehyde groups present in thepolysaccharide.

Also DE-198 21 859-A1 describes polysaccharide-antigen conjugates,wherein a suitable crosslinker is bound in the polysaccharide by meansof an azomethin bond to aldehyde functions obtained by periodateoxidation. In the cross-linker, a maleimido function is additionallyprovided, to which an —SH group of cysteine can add. The utilizedantigens then are N- or C-terminally provided with an additional Cys soas to allow for the addition of the terminal SH function with thecross-linker and thus the obtaining of the polysaccharide-antigenconjugates described.

Finally, U.S. Pat. No. 5,846,951 relates to polysaccharides comprisingat least 5 sialic acid residues which polysaccharides can be providedwith terminal aldehyde groups at the non-reducing ends of the polysialicacids by means of oxidation with sodium periodate. Terminal aldehydegroups created in this manner may then bind amino-group-containingmedicaments, e.g proteins, via azomethine bonds.

Most antigens used for vaccines comprise structures with primary aminogroups. In particular, all protein antigens normally comprise at leastone, but mostly several, lysines in their amino acid sequence. The aminogroups of these lysines are present in free form.

It has long been known that primary amines can react with aldehydes. Theproduct of this reaction is called Schiff's base. Schiff's bases are notcompletely stable compounds, they can be hydrolyzed under suitableconditions and thus be returned into their starting substances.

Furthermore, it has been known that compounds comprising vicinalhydroxyl groups can be oxidized with the help of suitable oxidants, inparticular with periodic acid or salts of periodic acid, such as sodiummetaperiodate, such that two aldehyde functions are formed by breakingthe C—C bond on which the neighboring hydroxyl groups are located.

A large number of high-molecular polysaccharides consist of monomericsugar units which carry vicinal hydroxyl groups. Dextrane and mannanshould be mentioned as two non-limiting examples. Such polysaccharidesthus can be oxidized with periodate in the above-described mannerwithout the bonds between the monomers being split. If, based on thenumber of monomeric units, a stoichiometric smaller amount of periodateis used, the oxidation will occur only partially, which means that onlyso many monomers will be oxidized according to the principle of randomas corresponds to the amount of periodate.

The present invention is based on the object of providing further meansand methods which will lead to immunogenic formulations of vaccines.

In a method of the initially defined type, this object is achieved inthat a polysaccharide carrier comprising vicinal hydroxyl groups isoxidized under ring opening to create vicinal aldehyde groups, and isreacted with one or several base-instable antigenic polypeptide(s)containing at least one free amino group, wherein the polypeptide(s) is(are) bound directly to the polysaccharide carrier via at least oneazomethine bond. Partially oxidized polysaccharides thus are a suitablecarrier material for the formulation of vaccines if the utilizedbase-instable antigenic polypeptides comprise one or more free primaryamino groups and thus, via an azomethine bond, can be connected with thevicinal aldehyde groups created in the carrier material by ring opening.Preferably, the base-instable antigenic polypeptides used according tothe invention are stable up to a pH of approximately 11, preferably upto a pH of approximately 10, still more preferred up to a pH ofapproximately 9, most preferred up to a pH of approximately 8. Ifpolypeptides are mentioned in the context of the present invention,proteins having at least 6 amino acids in the chain are to beunderstood. In the same way, polysaccharides are understood to bepoly-sugars comprising at least 3 monomer units in the chain. Preferablyused polysaccharides are mannan, e.g. having a molecular weight of atleast 70 kDa, and dextrane, e.g. having a molecular weight of at least70 kDa, particularly preferred having a molecular weight ofapproximately 2000 kDa.

According to a preferred embodiment of the present invention, thevicinal hydroxyl groups originally present in the polysaccharide carrierare at least partially oxidized, preferably by at least 20%. Bycontrolling the rate of oxidation, e.g. by a stoichiometric smalleramount of oxidating agent, the amount of aldehyde groups available foran azomethine bond between carrier and polypeptide can easily beadjusted.

Preferably, the base-instable antigenic polypeptide is a vaccineantigen, particularly preferred an antibody, e.g. a monoclonal antibody,such as the murine monoclonal antibody HE2. A new method of cancervaccination has been described in application PCT/EP00/00174 (prioritydate: Jan. 13, 1999), “Verwendung von Antikörpern zur Vakzinierung gegenKrebs” (“The Use of Antibodies for Vaccinating against Cancer”), thedisclosure of which is included herein by reference thereto. Themonoclonal antibody HE2 described there which is used as the vaccineantigen in a cancer vaccination serves as a non-limiting example for theformulation of a vaccine according to the method of conjugation to apartially oxidized high-molecular polysaccharide described here.

According to a further preferred embodiment of the present invention,the base-instable antigenic polypeptide has the same binding finespecificity as the antibody HE2.

It is also suitable if in addition to the respective base-instableantigenic polypeptide substances are conjugated which cause anenhancement of the immune response, e.g. GM-CSF, IL-2, IL-12 orGamma-Interferon, or a mixture of these substances.

Moreover, it is preferred if the polysaccharide-polypeptide conjugateaccording to the invention is additionally adsorbed on aluminumhydroxide and/or mixed with pharmaceutically acceptable carriers.

Finally, it is preferred if the polysaccharide-polypeptide conjugateobtained according to the invention is formulated as a vaccineformulation to be administered by subcutaneous, intradermal orintramuscular injection, e.g. by dissolving or suspending theoptionally, e.g., aluminum-hydroxide-adsorbed conjugate in a suitablephysiological buffer and the like.

In general, the following advantages and specific properties of theconjugate according to the invention should be mentioned:

-   -   The components coupled to the oxidized polysaccharides via        primary amines (conjugate and adjuvants and additives,        respectively) are slowly released in the presence of an excess        of molecules with free primary amines, e.g. serum proteins. The        slow release effect thus forming is desired for vaccines, since        by this antigen-presenting cells are able to locally receive the        vaccination antigens at the site of vaccination for a longer        period of time.    -   By the choice of the polysaccharide, the properties of the        conjugate can be influenced. This applies both to the molecular        size of the polysaccharide and to its chemical composition. If,        e.g., mannan is chosen as the polysaccharide, the corresponding        conjugate preferably will be taken up by cells of the immune        system which carry the mannose receptor. Among them are, in        particular, macrophages and dendritic cells as professional        antigen-presenting cells. In this manner, an increased immune        response is attained.    -   Several components can simultaneously be bound to partially        oxidized polysaccharides. These may be several differing vaccine        antigens, or vaccine antigens together with components enhancing        the immune response, such as, e.g., the proteins GM-CSF, IL-2,        IL-12 or gamma interferon.

The enclosed FIG. 1 shows the comparison of two formulations as regardsthe induction of antibodies against HE2 (ELISA) in rhesus monkeys.

EXAMPLE

At first, dextrane having a molecular weight of 2000 kDa (SIGMA D-5376)is oxidized by 20% with sodium metaperiodate. For this purpose, 324 mgof dextrane are dissolved with stirring in 4 ml of distilled water. Tothis solution, 86 mg of sodium metaperiodate previously dissolved in 0.6ml of distilled water are admixed, and incubated in the dark at 37° C.for 30 minutes. 25 mg of the antibody HE2 (PCT/EP00/00174) are broughtto pH 7.4 with 1 M Na₂HPO₄, and 45 μl of a thimerosal solution (10mg/ml) are added.

To this solution, 1.675 l of the above-obtained oxidized dextranesolution are added and incubated in the dark at 37° C. for 2 days. Thecompleteness of the reaction is analytically checked by chromatographyon a molecular weight column (zorbax 450). The signal corresponding to amolecular weight of 150 kDa (monomeric HE2) has disappeared, and in itsplace a signal occurs in the exclusion volume of the column whichcorresponds to a molecular weight of >2000 kDa.

The solution obtained is chromatographed by means of a preparativemolecular weight column which is equilibrated with the final buffer (1mM phosphate buffer in physiological saline, pH=5.5). The materialobtained in the exclusion volume consists of the high-molecularconjugate of the antibody HE2 on partially oxidized dextrane. Thecontent of conjugated HE2 can be determined by integration of the signalafter analytical chromatography on a molecular weight column as comparedto monomeric HE2. The solution obtained is mixed with an aqueousaluminum hydroxide such that the final concentration is 0.5 mg of HE2 on1.67 mg of aluminum hydroxide in 0.5 ml of buffer.

Four rhesus monkeys are subcutaneously immunized with 0.5 ml of theabove formulation on days 1, 15, 29 and 57. The sera of various pointsof time were assayed by means of ELISA for an induction of antibodiesagainst monomeric HE2. As a comparison, four rhesus monkeys werevaccinated in the same manner with a standard formulation of 0.5 mg ofmonomeric HE2 adsorbed on 1.67 mg of aluminum hydroxide.

The ELISA was carried out as follows:

100 μl aliquots of the MAb HE2 (solution with 10 μg/ml in bindingbuffer) are incubated in the wells of a microtiter plate for 1 h at 37°C. After having washed the plate six times with washing buffer A, 200 μleach of blocking buffer A are added and incubated at 37° C. for 30minutes. After washing the plate as described above, 100 μl aliquotseach of the monkey sera to be tested are incubated in dilutions of 1:100to 1:1 000 000 in dilution buffer A at 37° C. for 1 h. After havingwashed the plate as described above, 100 μl each of theperoxidase-conjugated goat anti-human-Ig antibody (Zymed) is added in adilution of 1:1000 in dilution buffer A and incubated at 37° C. for 30minutes. The plate is washed four times with washing buffer A, and twicewith staining buffer. The antibody bond is detected by the addition of100 μl each of the specific substrate, and the staining reaction isstopped after 10 minutes by adding 50 μl each of stopping solution. Theevaluation is effected by measuring the optical density (OD) at 490 nm(wave length of the reference measurement is 620 nm).

The results of the ELISA are illustrated in FIG. 1. The animalsvaccinated with the conjugate of HE2 on dextrane developed comparabletiters of antibodies against HE2 as the monkeys vaccinated with thestandard formulation.

Moreover, it was investigated to which extent HE2 after application canbe found again in the serum of the monkeys. For this purpose, again anELISA was used, which was carried out as follows:

100 μl aliquots of a purified polyclonal anti-idiotypic goat antibodyagainst HE2 (solution with 10 μg/ml in binding buffer) are incubated inthe wells of a microtiter plate at 37° C. for 1 h. After having washedthe plate six times with washing buffer A, 200 μl each of the blockingbuffer are admixed and incubated at 37° C. for 30 minutes. After havingwashed the plate as described above, 100 μl aliquots each of the monkeysera to be tested are incubated in dilutions of 1:4 to 1:100 000 indilution buffer A at 37° C. for 1 h. After having washed the plate asdescribed above, 100 μl each of a peroxidase-conjugated goatanti-mouse-IgG antibody (Zymed) are added in a dilution of 1:1000 indiluting buffer and incubated at 37° C. for 30 min. The plate is washedfour times with washing buffer, and twice with staining buffer. Theantibody bond is detected by the addition of 100 μl each of the specificsubstrate, and the staining reaction is stopped after 10 minutes byadding 50 μl each of stopping solution. The evaluation is effected bymeasuring the optical density (OD) at 490 nm (wave length of thereference measurement is 620 nm).

the results are illustrated in the following table:

Time Conjugate vaccine Standard formulation  0 0; 0; 0; 0 ng/ml  0; 0;0; 0 ng/ml  1 h 0; 0; 0; 0 ng/ml  13; 17; 74; 280 ng/ml  4 h 0; 0; 0; 0ng/ml 200, 280, 400, 740 ng/ml 24 h 0; 0; 0; 0 ng/ml 960, 960, 1000, 740ng/ml

After 24 h, a trace of a HE2 concentration can be recognized in theserum of those animals which had been vaccinated with the conjugate, yetthis concentration is below the detection limit of approximately 10 ngof HE2/ml serum. Apparently, the desorption of the vaccine antigen hasclearly been reduced by the conjugation to partially oxidized dextrane,as compared to the standard formulation. Thereby probably fewervaccinations will suffice to obtain a comparable titer than with astandard formulation on aluminum hydroxide.

Materials used: Microtiter Immuno Plate F96 MaxiSorp (Nunc) for plates:ELISA Binding buffer:   15 mM Na₂CO₃   35 mM NaHCO₃   3 mM NaN₃ pH: 9.6PBS deficient:  138 mM NaCl  1.5 mM KH₂PO₄  2.7 mM KCl  6.5 mM Na₂HPO₄pH: 7.2 Washing buffer: 0.05% Tween 20 in PBS deficient Blocking buffer:  5% fetal calf serum (heat-inactivated) in PBS deficient Dilutionbuffer:   2% fetal calf serum (heat-inactivated) in PBS deficientStaining buffer: 24.3 mM citric acid 51.4 mM Na₂HPO₄ pH: 5.0 Substrate:  40 mg o-phenylene-diamine-dihydrochlo- ride  100 ml staining buffer  20 μl H₂O₂ (30%) Stopping solu-   4 N H₂SO₄ tion:

1. A method of producing a polysaccharide-polypeptide conjugate,comprising: providing a polysaccharide carrier having vicinal hydroxylgroups, providing at least one antigenic polypeptide having at least onefree amino group, partially oxidizing said polysaccharide carrier underring opening so as to form vicinal aldehyde groups, reacting saidoxidized polysaccharide carrier with said at least one antigenicpolypeptide having the at least one free amino group, said at least oneantigenic polypeptide being bound directly to said polysaccharidecarrier via at least one azomethin bond thereby forming saidpolysaccharide-polypeptide conjugate.
 2. The method of claim 1, whereinsaid partially oxidized polysaccharide carrier is oxidized by at least20%.
 3. The method of claim 1, wherein said at least one antigenicpolypeptide is stable up to a pH of approximately
 11. 4. The method ofclaim 1, wherein said at least one antigenic polypeptide is stable up toa pH of approximately
 10. 5. The method of claim 1, wherein said atleast one antigenic polypeptide is stable up to a pH of approximately 9.6. The method of claim 1, wherein said at least one antigenicpolypeptide is stable up to a pH of approximately
 8. 7. The method ofclaim 1, wherein said polysaccharide is mannan.
 8. The method of claim7, wherein said mannan has a molecular weight of at least 70 kDa.
 9. Themethod of claim 1, wherein said polysaccharide is dextrane.
 10. Themethod of claim 9, wherein said dextrane has a molecular weight of atleast 70 kDa.
 11. The method of claim 9, wherein said dextrane has amolecular weight of approximately 2000 kDa.
 12. The method of claim 1,wherein said antigenic polypeptide is a vaccine antigen.
 13. The methodof claim 12, wherein said vaccine antigen is an antibody.
 14. The methodof claim 13, wherein said antibody is a monoclonal antibody.
 15. Themethod of claim 14, wherein said monoclonal antibody is a murinemonoclonal antibody (HE2).
 16. The method of claim 13, wherein saidantibody has a binding fine specificity equal to the binding finespecificity of a murine monoclonal antibody (HE2).
 17. The method ofclaim 1, further comprising providing immune-response-enhancingsubstances, and additionally conjugating said immune-response-enhancingsubstances to said antigenic polypeptide; wherein saidimmune-response-enhancing substances are selected from the groupconsisting of granulocyte macrophage colony-stimulating factor (GM-CFS),IL-2, IL-12, gamma interferon and mixtures thereof.
 18. The method ofclaim 1, further comprising providing aluminum hydroxide and adsorbingsaid polysaccharide-polypeptide conjugate on said aluminum hydroxide.19. The method of claim 1, further comprising providing apharmaceutically acceptable carrier and mixing said formedpolysaccharide-polypeptide conjugate with said pharmaceuticallyacceptable carrier.